This application will address NCI Provocative Question 11: How do changes in RNA processing contribute to tumor development? Tumors and leukemias have dramatically increased levels of aberrant RNA splicing, which generates a large population of transcripts that could encode variant proteins. This increased complexity of RNA products could be due to noisy splicing that is increased in transformed cells, but has no functional consequence. Alternatively, the variant RNAs and the proteins they encode could actively contribute to the transformed phenotype. Increased levels of alternative splicing in tumors leads to the expression of transcripts that are too numerous to test individually, for example by over-expression or knock down experiments. Furthermore, if the importance of the variant proteins is due to a mass action or combinatorial effect, then assessing their importance individually will not suffice. Our hypothesis is that if the increased alternative splicing exhibitd by tumors or leukemias contributes to tumor development, then determining the predicted expression levels of protein variants encoded by the alternatively spliced RNAs will be more informative, and will correlate with outcome better than simply measuring the RNA levels. If the products of alternative splicing play no significant biological role, then determining which proteins are produced by the alternatively spliced RNAs will offer no additional advantage in predicting outcome. We will apply an innovative next-generation RNA sequencing approach to the analysis of alternative RNA splicing in a large group of high risk childhood B- progenitor Acute Lymphocytic Leukemia (ALL) samples. Despite recent improvements in the treatments for B- ALL, this high risk cohort represents a group of patients for whom few good treatment options exist. Our approach will produce structural information over the entire length of more than 99% of expressed transcripts, allowing us to analyze gene expression, RNA splicing and the populations of protein variants that are predicted to be produced in each sample. Comparing these data sets will allow us to answer the Provocative Question and to determine whether increased levels of alternative RNA splicing are important in the development of B- progenitor ALL. The alternative splicing machinery contains many poorly characterized enzymes and regulatory proteins. If increased alternative splicing is found to play a role in tumor development these proteins will represent novel potential targets for the development of new drugs or interventions. PUBLIC HEALTH RELEVANCE: Although optimized treatment strategies have improved the survival rates for most children with B- progenitor Acute Lymphocytic Leukemia (B-ALL), there remains a group of high risk patients who fail standard treatments and have few good therapeutic options. We propose to study a complicated and poorly understood process, alternative RNA splicing, in a large group of samples from children with high risk B-ALL to determine whether aberrant regulation of RNA splicing contributes to the disease. If aberrant alternative splicing is involved, new targets could be identified for the development of novel drugs or therapeutic strategies.